1. Field of the Invention
The present invention provides for novel method for producing novel chimeric molecules that have enhanced biological activity, and easier production and purification processes as compared with the molecules in their native form. The invention also provides for nucleic acids useful for producing biologically active chimeric polypeptides, and the fusion polypeptides themselves.
2. Description of the Background
Transmembrane protein kinases serve as signaling receptors for a variety of polypeptide ligands, eliciting such diverse responses as cell survival, proliferation and differentiation from many cell types and tissues (van der Geer et al., 1994, Annu Rev Cell Biol. 10:251-337). Receptor tyrosine kinases (RTKs) have the ability to interact with different ligands and bring about various cellular responses. One type of RTK is tyrosine kinase with immunoglobulin and epidermal growth factor receptor homology domains (Tie), Tie2 (Dumont et al., 1993, Oncogene 8:1293-1301; Mustonen and Alitalo, 1995, J. Cell Biol. 129:895-898). Tie2 is expressed predominantly on endothelial cells, hematopoietic cells, or their embryonic precursors, and it is required for normal vascular development (Sato et al., 1995, Nature 376:70-74). Functional disruption of Tie2 in transgenic mice results in embryonic lethality by day E9.5 to 10.5, with effects on the microvasculature resulting in reduced numbers of endothelial cells, and abnormalities of vascular morphogenesis and hematopoiesis (Sato et al., 1995, Nature 376:70-74). Thus, Tie2 is critical for angiogenesis and hematopoiesis during development.
Davis et al. discovered that Ang1 is the ligand for Tie2 (Davis et al., 1996, Cell 87:1161-1169; WO9611269) (FIG. 1). Ang1 contains 498 amino acids, including an amino-terminal secretory signal sequence (FIG. 1). Human and mouse Ang1 are 97.6% identical. The amino-terminal region, consisting of residues 100-280, is weakly related to myosin and its relatives, in the regions of these proteins where they are known to possess coiled-coil quaternary structure (FIG. 1). The second region, consisting of residues 280-498, has strong similarity to the carboxy-terminal domain of fibrinogen (FIG. 1). Ang1 is a multimer, held together by coiled-coil structures and disulfide crosslinks. Recombinant Ang1 is a 70-kDa (reduced condition) secreted glycoprotein that binds to the Tie2 receptor with a Kd of approximately 3.7 nM, and induces tyrosine phosphorylation of Tie2 in endothelial cells (Davis et al., 1996, Cell 87:1161-1169).
Angiopoietin-1 (Ang1) is a specific and critical growth factor for blood vessel formation (Davis et al., 1996, Cell 87:1161-1169; Yancopoulos et al., 2000, Nature 407:242-248). Recent studies indicate that Ang1 could be used for preventing vascular leakages, therapeutic vasculogenesis, and therapeutic endothelial cell survival (Thurston et al., 2000, Nat. Med. 6:460-463; Chae et al., 2000, Arterioscler. Thromb. Vasc. Biol. 20:2573-2578; Kwak et al., 2000, Circulation 101:2317-2324). However, Ang1 protein is not easily available, and generation of recombinant Ang1 is extremely difficult with current techniques. Multimerization of the coiled coil domains during production of the ligand hampered purification.
Complementary DNAs encoding angiopoietin-2 (Ang2) were isolated by low-stringency screening of a cDNA library by using Ang1 cDNA as a probe (Maisonpierre et al., 1997, Science 277:55-60). Ang2 contains 496 amino acids and has a secretory signal sequence. Human and mouse Ang2 are 85% identical and approximately 60% identical to Ang1. Like Ang1, Ang2 has an amino-terminal coiled-coil domain and a carboxy-terminal fibrinogen-like domain. Ang1 and Ang2 have similar binding affinities for Tie2. Ang2 acts as an antagonist of Tie2 through inhibition of Ang1-induced phosphorylation of Tie2 (Maisonpierre et al., 1997, Science 277:55-60). Mouse angiopoietin-3 (Ang3) and human angiopoietin-4 (Ang4) were identified through low stringency hybridization screening with Ang1 and Ang2 cDNAs (Valenzuela et al., 1999, Proc Natl Acad. Sci. 96:1904-1909). Ang3 and Ang4 are probably interspecies orthologs. Ang4 phosphorylates Tie2, while Ang3 inhibits Ang1-induced phosphorylation of Tie2 (Valenzuela et al., 1999, Proc Natl Acad. Sci. 96:1904-1909).
Multimeric form of Ang1 phosphorylates Tie2. In turn, phosphorylated Tie2 interacts cytoplasmically with Grb2, Grb7, Grb14, the protein tyrosine phosphatase Shp2, and the p85 subunit of phosphatidylinositol 3′-kinase (PI 3′-kinase) via their SH2 domains (Jones et al., 1999, J Biol. Chem. 274:30896-30905). Association between p85 and Tie2 results in PI 3′-kinase activation and subsequent induction of the serine-threonine kinase Akt. (Kontos et al., 1998, Mol Cell Biol. 18:4131-4140). Ang1 induces endothelial cell survival through this PI 3′-kinase/Akt signaling pathway (Kim et al., 2000, Circ Res. 86:24-29). In addition, Ang1 induces endothelial cell sprouting through the activation of PI 3′-kinase and focal adhesion kinase (Kim et al., 2000, Circ Res. 86:952-959). Thus, Tie2, PI 3′-kinase, Akt, and focal adhesion kinase are crucial elements in the signal transduction pathway leading to survival and migration in endothelial cells. Phosphorylated Tie2 also interacts with Dok-R/Dok-2, leading to activation of Dok-R/Dok-2 (Jones and Dumont, 1998, 17:1097-1108). Phosphorylated Dok-R interacts with rasGAP, Nck, and Crk (Jones and Dumont, 1998, 17:1097-1108). These signaling molecules may be involved in cell migration and proliferation, organization of the cytoskeleton, and regulation of Ras signaling. Recently, it was shown that Dok-R/Dok-2 is responsible for recruiting Nck and p21-activating kinase (Pak/Pak1) to the activated receptor (Master et al., 2001, EMBO J. 20:5919-5928). Localization of this Dok-R-Nck-Pak complex to the activated Tie2 at the cellular membrane is coincident with activation of Pak kinase (Master et al., 2001, EMBO J. 20:5919-5928). This signal transduction pathway may be involved in Ang1-mediated migration in endothelial cells. Signal transducers and activators of transcription (STATs) were also found to be potential targets of Tie2 activation (Korpelainen et al., 1999, Oncogene, 18:1-8). Phosphorylated Tie2, in turn, activates STAT3 and STAT5 (Korpelainen et al., 1999, Oncogene, 18:1-8). Since STAT3 and/or STAT5 are known to be involved in the regulation of cell proliferation, differentiation, migration, and survival in many biological systems, it is possible that some of the Tie2 functions in endothelial cells may be controlled through STAT pathway.
It is known that the Tie2 receptor is expressed not only in endothelial cells but also in hematopoietic stem cells (HSCs), indicating another possible role of Ang1 and Tie2 in hematopoiesis (Iwama et al., 1993, Biochem Biophys Res Commun 195:301-309). In fact, Tie2 deficient mice show severely impaired hematopoiesis (Sato et al., 1995, Nature 376:70-74). And HSCs closely adhere to endothelial cells at several sites in the embryo. Furthermore, it has been found that HSCs produce Ang1, suggesting that HSCs can promote the migration of endothelial cells and establish a hematopoietic environment (Takakura et al., 2000, Cell 102:199-209).
Ang1*, made by Regeneron Pharmaceuticals, Inc., is a recombinant version of Ang1 that is easier to produce and purify (Maisonpierre et al., 1997, Science 277:55-60; (PCT WO 98/05779)). Ang1* contains a modified amino-terminus and mutated Cys265. The biological activity of recombinant Ang1 and Ang1* is similar. However, some of the same problems are encountered in producing Ang1* because the size of Ang1* is too large for efficient recombinant generation.
Both native Ang1 and Ang1* require extensive, expensive and labor-intensive purification schemes that result in relatively poor yields of recombinant protein. The need for cost-effective, simple purification schemes for biologicals intended for clinical use cannot be over-emphasized.
U.S. Pat. No. 6,455,035 discloses a method of decreasing or inhibiting vascular permeability in a mammal by administering to the mammal a Tie-2 receptor activator. However, U.S. Pat. No. '035 also discloses that the coiled coil domain of Ang1 was deleted so that the multimerization of the coiled coil domains would not hamper ligand purification.
WO 00/37462 discloses a method of enhancing the biological activity of Ang1 through deletion of coiled-coil domain and insertion with Fc-portion of immunoglobulin for making a tetramer form of chimeric fusion Ang1 (Ang1-1FD-Fc-FD). However, WO 00/37462 discloses that Ang1-1FD-Fc-FD is equivalent to Ang1* in its ability to stimulate phosphorylation of the Tie2 receptor.
Therefore, there is a need in the art to make a modified ligand molecule that is soluble and easily produced, while having substantially similar or greater potency as the native molecule.